NOTCH1 was discovered through analysis of a (7;9) chromosomal translocation found in <1% of T-ALLs, which creates a truncated NOTCH1 gene encoding constitutively active ICN1-like polypeptides. Prior work (from us and others) has shown that constitutively active forms of NOTCH 1 are potent inducers of T cell acute lymphoblastic leukemia/lymphoma (T-ALL) in mice. Recently, we identified frequent gain of function NOTCH1 mutations in human T-ALL cell lines and primary tumors. Two different types of mutations occur: point mutations involving a heterodimerization domain lying just external to the transmembrane domain;and frameshift or stop codon mutations that result in deletion of the cytoplasmic tail of NOTCH 1. These mutations activate NOTCH1 signaling synergistically, probably by increasing both the production and stability of ICN1. Beyond providing important pathogenic insights, these findings have exciting therapeutic implications; because NOTCH1 receptors bearing these mutations depend on the protease gamma-secretase for function, these molecular lesions can be targeted with gamma-secretase inhibitors. Based on these new insights, we propose the following three specific aims: 1. To determine how mutations in the heterodimerization domain of NOTCH1 increase signaling 2. To determine how mutations that delete the C-terminal sequences of NOTCH1 increase signaling 3. To determine the effects of leukemia-associated NOTCH1 mutations on human hematopoietic stem cell development and transformation Molecular models for how NOTCH1 mutations produce increases in downstream signaling will be tested, initially in cell-based assays and subsequently in a robust murine leukemia model. Because human cells may have different requirements for transformation than murine cells, we will explore the effects of various mutated forms of NOTCH1 on primary human hematopoietic progenitors. Together, these studies will yield important new insights into how NOTCH1 contributes to human acute leukemias. Because the NOTCH pathway is a tractable therapeutic target, these insights are likely to yield new therapeutic opportunities in acute leukemia and other cancers associated with abnormalities in NOTCH signaling.